Tuberous sclerosis complex (TSC) is caused by germline inactivating mutations in the TSC1 or TSC2 genes, which encode hamartin (TSC1) and tuberin (TSC2), respectively. Both humans and mice with mutations in TSC1 or TSC2 can develop cystic kidney disease. The TSC1 and TSC2 proteins function as a heterodimer to inhibit Rheb, which is the target of tuberin's GTPase activating domain. Rheb activates the mammalian target of rapamycin (mTOR) complex 1 (TORC1). We and others have observed evidence of mTOR pathway hyperactivation in cysts from autosomal dominant polycystic kidney disease (ADPKD) patients, suggesting that the TSC/Rheb/mTOR pathway is involved in ADPKD pathogenesis. In addition to the mTOR activation in ADPKD cysts and in cysts from mice with Pkd1 inactivation, two other lines of evidence suggest that the TSC proteins and Rheb are closely linked with the pathogenesis of ADPKD: the TSC2 protein has been found to regulate membrane localization of Polycystin 1 (PC1), and the overexpressed carboxyl-terminus of PC1 has been found to co-immunoprecipitate with TSC2. These data have led to the hypothesis that inactivation of PC1 is sufficient to activate mTOR. However, we have found that Pkd1-/- MEFs do not have evidence of mTOR activation and that mTOR is hyperactive in only a subset of ADPKD cysts. Elucidating the incompletely understood mechanisms through which mTOR is activated in a subset of ADPKD cysts, which is the focus of Aim 1, has particular clinical importance since rapamycin and other TORC1 inhibitors are now in clinical trials for patients with ADPKD. In unpublished work, we have found that hamartin (TSC1) localizes to the basal body of the primary cilium, and that Tsc1-null and Tsc2-null mouse embryonic fibroblasts (MEFs) have a higher fraction of ciliated cells than control MEFs. These data, which provide the foundation for Aim 2, link the TSC pathway with the cilium for the first time and suggest that Rheb may impact cyst pathogenesis through multiple mechanisms Our central hypothesis is that activation of Rheb cooperates with mutational inactivation of PKD1 to promote cyst pathogenesis in ADPKD. To test this, we propose the following Aims: Aim 1: To define the mechanisms through which mTOR is activated in ADPKD. Aim 2: To identify the mechanisms through which the TSC proteins regulate ciliary formation. Aim 3: To determine whether Rheb activation potentiates Pkd1-induced cyst pathogenesis. PUBLIC HEALTH RELEVANCE: Determining the mechanisms of mTOR activation in ADPKD and the role of the TSC proteins and Rheb in ciliary formation may have key therapeutic implications for the estimated 4-6 million ADPKD patients worldwide, particularly since mTOR inhibitors are already in clinical trials for PKD.